1. Field of the Invention
This invention relates to a method and apparatus utilizing the photothermal deflection effect to measure light absorption on a thin layer chromatographic plate. More specifically, the invention relates to the use of a pair of intersecting laser beams, one impinging on and the other parallel to the chromatographic plate, for quantitative determination of the chromatographically resolved compounds (thin layer, electrophoresis, etc.).
2. Description of the Prior Art
Thin layer chromatograph (TLC) is generally acknowledged as a simple, rapid and versatile separation technique. In recent years, TLC technology has benefitted from the introduction of "high performance" plates, coated with very uniform layers of 5 to 10.mu. diameter silica. However, this high performance thin layer chromatography (HPTLC) requires both improved sample introduction techniques and improved detections systems. For example, in conventional thin layer chromatography, developed spot sizes are usually 5 to 6 mm in diameter. However, in HPTLC the spot size is only 1 to 2 mm in diameter and further reduction in spot size is possible. Consequently, the need still exists for detectors which can probe areas of approximately one square millimeter or less and still meet or exceed the characteristics of known detection systems.
Although the principles of diffuse reflectance spectroscopy, perhaps the most popular TCL detection technology, are well known and available in commercial instruments, the limitations of this detection system are also well recognized. The detection limit for diffuse reflectance is typically in the low nanogram range and is to be considered for purposes of HPTLC as a relatively insensitive measurement technique. Fundamentally, the reason is that as the amount of compound present decreases, the reflected light approaches an upper limit. Thus, measurement of a small amount of material requires measuring the small difference between two large numbers. Fluorescence detection, the most common alternative to reflectance measurements, avoids this problem and is ten to one thousand times more sensitive than reflectance, depending on the compound. Thus detection limits are often in the 1 to 10 picogram range. However, fluorescence detection is limited to those compounds which fluoresce, or for which suitable fluorescent derivatives can be conveniently prepared, and only a small fraction of known compounds fluoresce. Photoacoustic spectroscopy (PAC) has been suggested as an alternative for detection of non-fluorescent compounds in TLC. Thus, it is known to place an open-ended PAS cell directly on a TLC plate as well as to use Fourier Transform infra-red (FTIR) PAS on the TLC plates with either gas-microphone or piezoeloctric transducer detection. The detection limit of photoacoustic spectroscopy on TLC plate has been reported to be in the range of low nanograms to high picograms.
In princple, the properties of lasers make them prime candidates for optical detectors in contemporary chromatographic applications. For example, laser induced fluorescence has been investigated as a high performance liguid chromatography detector system, but again the fact that most compounds are not fluorescent and have very low quantum efficiencies limits such applications. Recently, thermal lens spectroscopy has been proposed as an alternative to photoacoustic spectroscopy. In thermal lens spectroscopy the change in index of refraction of a sample caused by absorption of light and subsequent heat generation is measured. However, the conventional thermal lens technique is not directly applicable to opaque solids; but a thermal lens can be formed in a coupling gas, such as air over a solid sample, and then probed as the deflection of a low power laser. This technique is called photothermal deflection or the "mirage effect". Photothermal deflection has been used as the detection scheme for FTIR spectroscopy of solids, but to the best knowledge of the inventors the thermal lens or photothermal deflection effect, prior to the present invention, has never been employed in the quantitative determination of compounds separated on thin layer chromatographic plates.